Improved anticancer efficacy of methyl pyropheophorbide-a–incorporated solid lipid nanoparticles in photodynamic therapy

Photodynamic therapy (PDT) is a promising anticancer treatment because it is patient-friendly and non-invasive. Methyl pyropheophorbide-a (MPPa), one of the chlorin class photosensitizers, is a drug with poor aqueous solubility. The purpose of this study was to synthesize MPPa and develop MPPa-loaded solid lipid nanoparticles (SLNs) with improved solubility and PDT efficacy. The synthesized MPPa was confirmed 1H nuclear magnetic resonance (1H-NMR) spectroscopy and UV–Vis spectroscopy. MPPa was encapsulated in SLN via a hot homogenization with sonication. Particle characterization was performed using particle size and zeta potential measurements. The pharmacological effect of MPPa was evaluated using the 1,3-diphenylisobenzofuran (DPBF) assay and anti-cancer effect against HeLa and A549 cell lines. The particle size and zeta potential ranged from 231.37 to 424.07 nm and − 17.37 to − 24.20 mV, respectively. MPPa showed sustained release from MPPa-loaded SLNs. All formulations improved the photostability of MPPa. The DPBF assay showed that SLNs enhanced the 1O2 generation from MPPa. In the photocytotoxicity analysis, MPPa-loaded SLNs demonstrated cytotoxicity upon photoirradiation but not in the dark. The PDT efficacy of MPPa improved following its entrapment in SLNs. This observation suggests that MPPa-loaded SLNs are suitable for the enhanced permeability and retention effect. Together, these results demonstrate that the developed MPPa-loaded SLNs are promising candidates for cancer treatment using PDT.

The solvent that demonstrated the best characteristics for this method was MeOH. A standard stock solution was prepared by dissolving 2 mg of an accurate amount of MPPa in 20 mL of MeOH. The standard stock solution was diluted with MeOH to obtain final concentrations of 1-20 ppm, and fivepoint linearity was determined. Standard solutions of different concentrations were prepared. Calibration curves and concentration versus absorbance units were constructed for each drug.
The precision of the test method was determined by performing an assay with six replicates of samples at test concentrations, and the relative standard deviation (RSD) of the assay results was calculated.
To study the accuracy of the method, recovery studies were performed by adding a known quantity of the standard to the pre-analyzed sample. The recovery was performed at 0%, 25%, and 100% levels, and the contents were measured from the respective UV-Vis absorption spectra.
Determination of nanoparticle size, polydispersity index (PDI), and zeta potential for MPPa-loaded SLNs. The particle size and PDI of the prepared SLNs were determined at 25 °C by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, Malvern, UK) [31][32][33] . The zeta potential of SLNs was estimated from the electrophoretic mobility of the particle surface using a Zetasizer Nano ZS. The samples were diluted 10 times with distilled water (DW) before the measurement. The instrument was equilibrated before each measurement. Each value reported is the average of three measurements.
Determination of drug-loading capacity MPPa-loaded SLNs. The entrapment efficiency (EE) and loading amount (LA) of MPPa-loaded SLNs were determined by centrifugation 29 . SLN preparations were diluted 10 times to a final volume of 1 mL and then gently vortexed. The suspension was then centrifuged at 190 g at 4 °C for 1 h. The free drug concentration in the supernatant was analyzed using a UV-Vis spectrophotometer. EE and LA were calculated using Eqs. (1) and (2), respectively.
In vitro MPPa release studies. An in vitro MPPa release study was performed using the dialysis-bag method. Dialysis bags (Spectrum Laboratories, Inc., Compton, CA, USA) with a molecular weight of 10 kDa were soaked in DW for 12 h before the experiment 34 . A predetermined amount of each test substance was soaked in dialysis bags and both ends were sealed using a string. Dialysis bags were immersed in 70 mL vials containing 50 mL of receptor medium (PBS, pH 7.4). The vials were then placed in a shaking incubator (JSSI-100 T, JS Research Inc., Gongju, Korea) and shaken at 100 rpm and 37 ± 0.5 °C. At predetermined time intervals (1, 2, 4, 8, 12, 24 and 48 h), aliquots of 1 mL were withdrawn from the vial, passed through 0.45 μm membrane filters (SFCA Syringe Filters, Corning Inc., NY, USA), and immediately analyzed using a UV-Vis spectrophotometer.
The drug release kinetics models. To explain the mechanism of MPPa releases from the SLNs, the MPPa release profiles of the SLNs were analyzed with various models of release kinetics including zero-order, firstorder, Higuchi, and Korsmeyer-Peppas models using Eqs. (3), (4), (5) and (6), respectively.
where Q t is the amount of drug release at time t, Q 0 is the initial amount of drug in formulations, K 0 , K t , K H are release rate constants, C 0 is the initial concentration of drug.
Photostability studies. The photostability of MPPa in SLNs was determined by comparison with MPPa in 0.1% MeOH solution 29 . The photostability of MPPa was monitored by recording its absorption spectrum at 700 nm. Briefly, 20 mL of MPPa or MPPa-loaded SLNs in a 0.1% MeOH solution (4.0 ppm) was irradiated with a light-emitting diode (LED) at different time intervals (0, 10, 20, 30 and 40 min). MPPa was then extracted from the formulations by adding 1 mL hexane to melt the lipids, followed by vortexing. A 0.1% MeOH layer containing the extracted MPPa was filtered through 0.22 μm filters, and the UV-Vis spectrophotometer was measured.  (2) LA (%) = Amount of total drug content − Amount of free drug Amount of total drug content − Amount of free drug + Amount of lipid × 100.
(3) Q t = K 0 t + C 0 , In vitro photoirritation study using human tumor cells. The anticancer efficacy of PDT with MPPa was evaluated by investigating the cytotoxic effects of each component of SLNs in tumor cell lines after photoirradiation 29,34 . Two cell lines (HeLa from human cervical carcinoma and A549 from human lung carcinoma) were seeded into 48-well plates at 2 × 10 4 cells/well, and the number of cells was calculated using a hemocytometer. Prior to each experiment, the cells were incubated for 24 h at 37 ± 0.5 °C in a humidified atmosphere with 5% CO 2 . Various concentrations (1, 2.5, 5, and 10 μM) of each sample were then added to each well. After 24 h, the exposed cells were rinsed with sterile PBS and incubated with 200 µL/well of the growth medium. The cells were then irradiated (2 J/cm 2 ) with LED at a distance of 20 cm for 15 min. The treated cells were incubated for 24 h at 37 ± 0.5 °C and 5% CO 2 for the WST reduction experiment.
Viability of cancer cells. Cytotoxicity was determined by measuring the dehydrogenase activity of viable keratinocytes at 24 h after incubation. Activity was determined after the incorporation of WST, as previously described 37 . Each cell line was treated with 100 µL/well of a 10% WST solution for 1 h. The WST concentration was measured by determining the optical density (OD) at 450 nm using a microplate reader.
Each experiment was conducted in at least three wells of a plate. After subtracting the blank OD from all raw data, the mean OD values ± standard deviations (SDs) were calculated using three measurements per test substance, and the percentage of cell viability relative to that of the NC was expressed using Eq. (8). The NC value was set at 100%.

Statistical analysis.
Three independent experiments were performed for all analyses. The presented data (mean ± SD) were compared using a one-way analysis of variance and Student's t-test. Statistical significance was set at p < 0.05. . Our results revealed that the peak for -OCH 3 (13 4 H at 3.88 ppm as previously reported by us) in MPa disappeared after elimination of -COOCH 3 , and one proton signal of 13 2 at 5.13 ppm appeared that confirmed the formation of reduced five-membered ring in MPPa 32 .

Development of analytical method for MPPa.
We obtained absorption spectra using a UV-Vis spectrophotometer to determine the specific absorption wavelength of MPPa ( Fig. 2A). The specificity of MPPa was determined using MPPa standard stock solution, MPPa-loaded SLN, and placebo SLN (SLN without MPPa). The UV-Vis spectra of MPPa demonstrated that the maximum absorption wavelength was 667 nm. The placebo SLN had no interference spectra with that of MPPa. Therefore, we analyzed MPPa at 667 nm.
To calculate MPPa, we prepared a calibration curve using five standard stock solutions in a concentration range of 1-20 ppm. Figure 2B shows that the correlation coefficient obtained via the linear regression analysis was 1.
A precision study was performed to determine the closeness of the agreement for the same concentration with repeated measurements. The precision results are expressed as the RSD of repeatability. MPPa results demonstrated that the RSD (%) value of recovery was 1.06% (Table 2), which indicated the high precision of the proposed analytical method.
We determined the accuracy via the developed analysis of MPPa to determine the closeness of agreement between the test results and a conventional true or accepted reference value. The recovery of MPPa was calculated and expressed as RSD. The RSD (%) values resulting from the accuracy were 1.26%, 0.42%, and 0.36%, respectively, as shown in Table 3.
Particle characterization of MPPa-loaded SLNs for MPPa-loaded SLNs. Considering particle characterization, nanoparticle size, PDI, and zeta potential were determined to investigate the effects of different     www.nature.com/scientificreports/ lipids and surfactants. Particle size is an important parameter in cancer therapy and is based on the EPR effect as a passive targeting strategy 26,38 . For nanoparticles, the zeta potential is important because it indicates the electric surface potential on the particles that ensures particle stability 18,39 . The results of particle size, PDI, and zeta potential demonstrated that all the formulations ranged from 231.37 to 424.07 nm in size, had a PDI of 0.15 to 0.42, and zeta potential of − 17.37 to − 24.20 mV, respectively, as shown in Fig. 3A Considering the effects of lipid and surfactant concentrations on F3 and F6 to F10, the results of particle characterization demonstrated that an increase in the concentration of GMS as a lipid led to an increase in the particle size. For the surfactant, an increase in the concentration of PX 188 led to a decrease in the particle size. This observation suggests that the concentration effect of lipids is associated with the increased volume of the lipid matrix 23 . An increase in the concentration of surfactant causes the interface between O and W in the O/W emulsion to be effectively stabilized (increased zeta potential), and consequently reduces the particle size and enhances the particle stability 39 .

Determination of the drug-loading capacity. Loading capacity (EE and LA) is a significant parameter
in the formulation of lipid particle systems because it enhances the photostability of MPPa and avoids sideeffects in the human body. Figure 3C,D show the EE and LA of MPPa-loaded SLNs. The EE and LA of all formulations were 73.49-78.73% and 1.54-7.11%, respectively. Among F1, F2, and F3, which were prepared using different lipids, F3 prepared from GMS showed the greatest loading capacity. This observation suggests that longer chain fatty acids have a high affinity for MPPa [43][44][45] , as mentioned in the particle characterization section. Regarding the effect of surfactants, F3 using PX 188 had a high amount of EE among F3, F4, and F5. This suggests that the high HLB value of the surfactant affected the stable dispersion of the MPPa-dissolved O phase into the W phase 43,46,47 . Regarding the effect of lipid and surfactant concentrations, an increase in the concentration of both increased EE owing to an increase in the volume of the lipid matrix and particle stability.
In vitro MPPa release studies. The release profile of MPPa-loaded SLNs was determined using the dialysis membrane method. The cumulative percentage release of MPPa-loaded SLNs was in the order F1 > F2 > F5 > F4 > F3 > F6 > F7 > F8 > F9 > F10. Among the formulations of F1 to F6, this order was the same as the particle size and reverse of the order of zeta potential (low stability), as shown in Fig. 3A and B. Thus, the large particle size and low stability of SLNs induced high drug release from SLNs after 48 h. The MPPa release from SLN exhibited a sustained release profile, as shown in Fig. 4. The sustained release of MPPa was biphasic, with a relatively burst and delayed release. The relatively burst release of F1-F6 and F7-F10 was observed over 12 and 8 h, respectively, followed by a sustained release for 48 h. This observation suggests that the relatively burst release is attributed www.nature.com/scientificreports/ to the adhesive MPPa on the particle surface (shell), whereas the sustained release is owing to the MPPa encapsulated into the particle core 23 . Concerning the short burst release of F7-F10, formulations with a high loading capacity are encapsulated in the particle core rather than the shell, which leads to a delayed release 23 . In this sense, the order of MPPa release was exactly the reverse of the order of EE, as shown in Fig. 3C.
The drug release kinetics models. The MPPa release results were analyzed using release kinetics models (zero order, first order, Higuchi, and Korsmeyer-Peppas). Table 4 demonstrates that Higuchi model is the highest correlation coefficients (R 2 ) values. This suggests that MPPa was homogeneously loaded in entire lipid matrix of SLN. In this regard, MPPa release was dominated by diffusion and dissolution 48,49 . Photostability studies. In PDT, the photostability of the PS is important because it is closely related to its pharmacological effect. Our results revealed that all formulations improved the photostability of MPPa (Fig. 5).
The order of photostability after 40 min of irradiation was F10 > F9 > F8 > F7 > F6 > F3 > F4 > F5 > F2 > F1 > MPPa; this order was the exact reverse of that observed for the release profile of MPPa from SLNs shown in Fig. 4. The remaining concentration of MPPa from the MPPa solution was 57.97% and that from the formulations ranged from 74.67% to 91.43%. Thus, a solid lipid, as the main ingredient of SLN, physically prevents any interruption from environmental factors and maintains MPPa for a relatively long time 22,23,50 . The concentration effects of lipids and surfactants demonstrated that F7-F10 highly improved the photostability of MPPa probably because formulations with high loading capacity protect MPPa from environmental factors.  www.nature.com/scientificreports/ 1 O 2 photogeneration. The efficacy of PDT was investigated using the DPBF assay where DPBF reacts with 1 O 2 , which decreases the intensity of the DPBF absorption band 51,52 . We performed the DPBF assay with photoirradiation of the MPPa solution and all formulations to detect the generated 1 O 2 , as shown in Fig. 6A , which is the same as that observed for the release profiles in Fig. 4 and exactly opposite of the order reported for zeta potential and photostability in Figs. 3B and 5, respectively. Therefore, highly stable formulations afforded relatively low PDT efficacy among MPPa-loaded SLNs. The reason for this might be the environmental protective effect of the SLN system 22,23 .   Table 5. The order of PDT efficacy was the same as the order of zeta potential and the opposite of the order of particle size as following: Therefore, a small particle size with low aggregation (high zeta potential) results in better PDT efficiency. Regarding the effect of different lipids, F3 prepared using GMS showed high anticancer effects against both cell lines as compared with F1 prepared using PA. Moreover, although F7 and F9 had a higher MPPa loading capacity than F3, the PDT efficacy of F3 was higher than that of F7 and F9. Considering that the particle size of F3 was smaller than that of F7 and F9, the anticancer effects via PDT efficacy dominated the particle size rather than the drug-loading capacity 3,53,54 . In addition, the order of PDT efficacy was the same as that of release, except for F1. This is because the formulation with large particle size showed low zeta potential as a stability parameter that is affected by both high particle aggregation and fast drug release, as mentioned in the sections on particle size and drug release. Thus, F3 is a promising formulation for cancer treatment based on the EPR effect strategy among the tested substances.

Conclusion
In this study, we attempted to synthesize MPPa and fabricate MPPa-loaded SLNs as a promising cancer treatment for PDT to improve the photostability and pharmacological effects. 1 H-NMR results showed that all proton signals were assigned, indicating the successful synthesis of MPPa. All MPPa-loaded SLNs displayed highly enhanced photostability and 1 O 2 photogeneration compared with free MPPa. In terms of PDT efficacy, our SLNs showed better anticancer effects than free MPPa against HeLa and A549 cells. In addition, the cytotoxicity study was performed under dark and light conditions, which ensured that the normal state of MPPa and MPPa-loaded SLNs was safe unless otherwise irradiated. Among F1, F3, F7 and F9 SLNs, use of lipid with longer carbon chain (GMS) generated smaller particle sizes of SLNs. In addition, a decrease of the lipid (GMS) concentration (among F3, F7 and F9) is important to decrease the particle size which induced an increase of the stability (increase of the zeta potential). Finally, F3 SLNs displayed the highest PDT efficiency against both cell lines, which might be attributed to the smallest particle size as well as the highest stability and loading amount even though the lower 1 O 2 photogeneration and entrapment efficiency. Therefore, we can make a conclusion that the anticancer efficacy of MPPa-loaded SLNs was dominated by particle size and stability rather than entrapment efficiency. Thus, these results showed that MPPa-loaded SLNs are promising anticancer agents for PDT.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.